VOLUME 7 • NUMBER 3 • MARCH 2016 JOURNAL OF CLINICAL AND RESEARCHORIGINAL ARTICLE ARTICLE EXPERIMENTAL INVESTIGATIONS An easy-to-use, rapid and inexpensive method to determine methicillin resistance in Staphylococcus aureus Jülide Sedef GÖÇMEN1, Osman CAĞLAYAN2, Alpay AZAP3 1TOBB University of Economics and Tecnology, Faculty of Medicine, ABSTRACT Microbiology Department-Ankara/ Objective: We aimed to report a new turbidimetric method to identify methicillin resistance in TÜRKİYE 2Kırıkkale University, Faculty of S. aureus strains just two hours after identification of the microorganism, and to analyze Medicine, Biochemistry Department- diagnostic and discrimination abilities of this new method. Kırıkkale/TÜRKİYE Methods: A total of 319 S. aureus isolates were included. Identification of bacteria was done by 3Ankara University, Faculty of Medicine, the colony morphology, and conventional biochemical methods. The methicillin resistance of Infectious Diseases and Clinical the S. aureus strains was studied as indicated in Clinical Laboratory Standards Institute 2009. Bacteriology Department-Ankara/ The turbidimetric method we developed is based on different growth rates of S. aureus in two TÜRKİYE media, with or without oxacillin. The growth rates of MRSA and MSSA are similar in normal media, however the MRSA grows significantly faster in the media containing oxacillin. Therefore, after 2 hours of incubation, the difference of turbidity produced by bacteria is less in MRSA, and more in MSSA. The absorbance of the microplates were measured before incubation, and at 2nd and 3rd hours of incubation. The “absorbance rate” was calculated for each bacteria and the bacteria were classified as MRSA or MSSA based on the absorbance rate. Corresponding autor: Results: All MRSA and MSSA strains were correctly discriminated via our turbidimetric method, Julide Sedef Göçmen when an absorbance rate of 1.900 was taken as cut-off value. The new method could diagnose TOBB University of Economics and MRSA with 100% specificity and 100% sensitivity in just two hours. Tecnology, Faculty of Medicine, Conclusion: The turbidimetric method is a rapid, easy and cheap method that does not require Microbiology Department-Ankara/ any specific equipment. It can be easily performed in every microbiology laboratory. TÜRKİYE Key words: Turbidimetric method, MRSA, MSSA, methicillin resistance, Staphylococcus aureus E mail: [email protected] Tel no: +90 (312) 292 4431 Fax no: +90 (312) 292 4432 nosocomial infections. In staphylococci, INTRODUCTION methicillin resistance occurs due to production of PBP 2a instead of PBP 2 by a mutant Staphylococcus aureus, is a Gram positive chromosomal gene, mecA, and PBP 2a has lower bacteria with a high virulence, and it is isolated from humans as an infectious agent. Penicillins affinity for beta- lactam antibiotics. Resistant started to be used in 1945 to treat infections bacteria can keep on synthesizing their cell wall. caused by S. aureus, and penicillin resistance This protein makes MRSA strains resistant not occurred in a short time due to beta lactamase. only to methicillin, but also to all beta lactam 1-3 Currently, S. aureus isolates show a high resistance agents, and this causes a treatment challenge. 1 (95%) to penicillins. Until recently, MRSA caused only nosocomial Methicillin is a semi-synthetic penicillin infections; however recently, severe community- resistant to penicillinase, and it started to be acquired infections were observed in some used in 1960, and methicillin-resistant S. aureus groups (prisoners, sports teams, military units, Received: 29 June 2016, (MRSA) were isolated short after, in one year. homeless people etc.), particularly in the United Accepted: 29 September 2016 After 1980, the infections caused by MRSA States.1,3 The rates of catheter- related bacteriemia, DOI: 10.5799/jcei.328616 constituted a significant proportion of the ventilator-related pneumonia, and surgical site www.jceionline.org Copyright © JCEI / Journal of Clinical and Experimental Investigations 2016 | 239 Göçmen JS, et al. Easy way to determine MRSA and cutaneous infections caused by MRSA (10-50%) vary among of this suspension was inoculated to an area of 1 cm2 on the countries. This rate is 39.9% in Turkey.4 MRSA infections cause medium. The petri dishes were inoculated at 35 ºC for 24 hours. threefold increase in hospital stay (from an average of 4.5 days Presence of growth was evaluated at the end of this period.7 to 13.5 days), threefold increase in treatment cost, and fivefold 3. b. Determination of susceptibility with disk diffusion increase in mortality. The mortality rate due to invasiveS. aureus method: S.aureus inoculum was prepared according to 0.5 MF infections was reported as 19-34%.5 English medical data indicated turbidity standards. They were inoculated in Mueller Hinton that MRSA was responsible for 0.1% of all deaths, and 0.2% of Agar (Difco-USA) using three- dimensional inoculation method. in-hospital deaths between 2008 and 2012.6 Oxacillin (1 µgr) and cefoxitin (30 µgr) (BD-USA) antibiotic Owing to increasing frequency of community-acquired MRSA disks were placed onto the surface of the agar. The petri dishes strains, rapid diagnosis and rapid determination of antibiotic were incubated at 35 ºC for 18-24 hours. The diameters of the susceptibility, and starting appropriate treatment immediately susceptibility zones were measured. ATCC 25923 and MRSA become important not only in nosocomial, but also in community- ATCC 43300 strains were used as the control strains. The threshold acquired infections.1,3 Obtaining the result of the susceptibility zone diameters were regarded as ≤10 and ≥13 mm for oxacillin, test with routine methods (disk diffusion, microdilution, gradient and ≤21 and ≥22 mm for cefoxitin, and the strains were classified strips) takes 24 hours.7,8 as MSSA or MRSA.8 This time is quite long for patients in intensive care units, as 3.c. Determination of susceptibility with microdilution: well as the ones being treated in haematology and oncology The MIC values of all isolates for oxacillin and cefoxitin were clinics. Since urgent and appropriate therapy is essential in many determined using U-bottom microplates. Cation- adjusted Mueller situations, MRSA is targeted in daily practice when there is a Hinton broth was used as the medium. Dilutions of the isolates suspicion for a staphylococcal infection, and glycopeptide antibiotics were done to obtain the last bacterial concentration as 5x105 are administered empirically. However, those antibiotics cause bacteria/ml. The medium contained 2, 3, 4, 8 mg/L oxacillin more adverse effects. In addition, risk of failure is higher, and (Ox), or 4, 6, 8, 16 mg/L cefoxitin (Fox). Control of media, treatment response is delayed when compared to beta lactam antibiotic-added media, and the controls with standard strains antibiotics if the causative agent is MSSA. Therefore, obtaining were performed in all tests done in all strains. The microplates the result of the antibiotic susceptibility test is as important as were incubated at 35 ºC for 24 hours. The wells without any the determination of the causative agent in staphylococcal visible growth were determined at the end of this period.8 infections.1-3 4. Antibiotic susceptibility with turbidimetric method; In this study, we aimed to report a new turbidimetric method discrimination of MSSA and MRSA: 319 strains were analyzed to identify methicillin resistance in S. aureus strains just two in flat-bottomed microplates (12x8) using the method we developed. hours after identification of the microorganism, and to analyse 4.a . The medium (M) in turbidimetric method: A number diagnostic and discrimination abilities of this new method. of different liquid media were used including Mueller Hinton broth, Triptic Soy Broth, Cation- adjusted Mueller Hinton Broth, METHODS and BHIB in order to determine the medium to be used in the experiment. At the end, BHIB (Difco-USA) medium was decided. 1. BacteriaA total of 319 S. aureus isolates isolated from Twofold more dehydrated medium than indicated on the BHIB various samples were included in the study. The strains were package was used (2 x 185 = 370 gr/L), and a twice- concentrated kept in skim milk at -80 °C were passed into Triptic Soy Agar medium was obtained. It was autoclaved at 120 ºC for 15 minutes. and 5% Sheep Blood Agar plates, twice. The plates were incubated 4.b. The medium with antibiotic in turbidimetric method in the incubator for one night, at 35 °C.6 (oxacillin): A sterile, twice-concentrated BHIB medium containing 2. Identification of the bacteria: Identification of bacteria 8 mg/L oxacillin (O1002-Sigma-Aldrich-USA) was prepared. was done by the colony morphology and conventional biochemical 4.c. Bacteria in turbidimetric method: Comparative studies methods [catalase test, plasma coagulase test, and the effects on showed that 0.5 MF bacterial density used in the study was the mannitol in mannitol salt agar].6 best turbidity. To obtain this concentration after mixing, 1 MF 3. Determination of antibiotic susceptibility with classical (3 x 108 bacteria /ml) bacterial suspensions were prepared in method, discrimination of MSSA and MRSA: The methicillin normal saline for each isolate studied. resistance of the S. aureus strains was studied as indicated in 4.d. Pipetting in turbidimetric method: The experiment Clinical Laboratory Standards Instıtute (CLSI) 2009:7 was performed as follows: 3 wells for experiment (M with antibiotic 3.a. Oxacillin screening test: Muller Hinton agar medium + bacteria), and 3 wells for positive control (M + bacteria) (Table containing 6 microgram/L oxacillin (Sigma-USA) and 4% NaCl 1). The final concentration after pipetting was 185 gr/L for the were prepared. Bacterial colony suspension was equivelent to medium, 0.5 MF for bacteria suspensions, and 4 mg/L for oxacillin 0,5 McFarland standard. (MF) (1.5 x 108 bacteria/ml) and 10 µl (Table 1). 240 | Copyright © JCEI / Journal of Clinical and Experimental Investigations 2016 www.jceionline.org Göçmen JS, et al. Easy way to determine MRSA 4.e.